Anatomical adjustments of the tree hydraulic pathway decrease canopy conductance under long-term elevated CO2

Abstract The cause of reduced leaf-level transpiration under elevated CO2 remains largely elusive. Here, we assessed stomatal, hydraulic, and morphological adjustments in a long-term experiment on Aleppo pine (Pinus halepensis) seedlings germinated and grown for 22–40 months under elevated (eCO2; c. 860 ppm) or ambient (aCO2; c. 410 ppm) CO2. We assessed if eCO2-triggered reductions in canopy conductance (gc) alter the response to soil or atmospheric drought and are reversible or lasting due to anatomical adjustments by exposing eCO2 seedlings to decreasing [CO2]. To quantify underlying mechanisms, we analyzed leaf abscisic acid (ABA) level, stomatal and leaf morphology, xylem structure, hydraulic efficiency, and hydraulic safety. Effects of eCO2 manifested in a strong reduction in leaf-level gc (−55%) not caused by ABA and not reversible under low CO2 (c. 200 ppm). Stomatal development and size were unchanged, while stomatal density increased (+18%). An increased vein-to-epidermis distance (+65%) suggested a larger leaf resistance to water flow. This was supported by anatomical adjustments of branch xylem having smaller conduits (−8%) and lower conduit lumen fraction (−11%), which resulted in a lower specific conductivity (−19%) and leaf-specific conductivity (−34%). These adaptations to CO2 did not change stomatal sensitivity to soil or atmospheric drought, consistent with similar xylem safety thresholds. In summary, we found reductions of gc under elevated CO2 to be reflected in anatomical adjustments and decreases in hydraulic conductivity. As these water savings were largely annulled by increases in leaf biomass, we do not expect alleviation of drought stress in a high CO2 atmosphere.


Supplemental material to Gattmann et al. Plant Physiology
Supplemental Methods S1. Non-linear model fitting.
The following models were fit via Bayesian calibration as described in the main text. The number of accounted auto-correlation structures (number of seedlings measured) per model is given.

1) gc response to increasing vapor pressure deficit (VPD)
For both treatments (aCO2, n = 6 and eCO2 n = 6) we assumed gc to decline with VPD according to the following equation: (S1) in which gc is the canopy conductance in mol m -2 s -1 , VPD is the vapor pressure deficit in kPa, and a1 and b1 are the calibrated coefficients of the regression.
2) gc response to declining midday leaf water potential (Yleaf) For the two treatments (aCO2 and eCO2) we assumed a logistic decline of gc following Yleaf reductions, with a non-zero asymptote to represent minimum canopy conductance: in which 100 ⋅ * $ " $ ",$%& + is the percentage of gc with respect to the maximum canopy conductance, and Yleaf is the midday leaf water potential (MPa). Regarding the calibrated coefficients, a2 is the percent of stomatal conductance relative to the maximum at the asymptote, which is the equivalent of minimum gc.
b2 is the leaf water potential at which the percent of gc relative to maximum gc is , and c2 is a scaling factor.

3) ABA response to declining midday leaf water potential (Yleaf)
For the two treatments (aCO2 and eCO2) we assumed a potential increase of ABA concentration with declining Yleaf as follows: in which ABA is the concentration of abscisic acid in the leaves (ng g -1 ), Yleaf is the midday leaf water potential (MPa), and a4 and b4 are the calibrated coefficients for the regression.

4) Percent loss in conductance with declining xylem water potential (Yxylem)
For both treatments (aCO2 n = 5 and eCO2 n = 6) we fitted a cumulative probability function in form of a Weibull distribution: with PLC is the percent loss of hydraulic conductance (%),Yxylem is the xylem water potential (MPa), a5 and b5 are the calibrated coefficients, where a5 is a scale parameter of reference xylem water potential value (MPa), and b5 a shape parameter.  Table S2. Parameter estimates of the Bayesian models. Model coefficients are given per treatment (eCO2 = elevated CO2, aCO2 = ambient CO2) and during decreasing CO2 (from 900 to 400 to 200 ppm) in the eCO2 seedlings. All parameter values are reported as the median and the 95% credible intervals per treatment. Bold letters indicate nonoverlapping credible intervals between treatments for a given test. ABA is abscisic acid concentration (ng g -1 ), Yleaf is midday leaf water potential (MPa), PLC is percent loss in xylem hydraulic conductance (%). Yxylem is xylem water potential (MPa), gc is canopy conductance (mol m -2 s -1 ), PAR is photosynthetic active radiation (µmol m -2 s -1 ), VPD is vapor pressure deficit (kPa) and gc,rel is canopy conductance relative to the treatment-specific maximum canopy conductance (%). Supplemental Figure S1. Leaf-level gas exchange. Diurnal course of leaf-level transpiration (E) (a) and canopy conductance (gc) (b) as well as photosynthetic active radiation (PAR) (c) and vapor pressure deficit (VPD) (d) under ambient (aCO2) and elevated [CO2] (eCO2). Shown are quarter-hourly treatment means over three (aCO2) and four (eCO2) days of acclimation with shaded areas depicting ± standard deviation (n = 6 seedling per treatment). Supplemental Figure S3. Tree-level transpiration and photosynthesis. Diurnal course of tree-level transpiration (E) (a) and tree-level net photosynthesis (Anet) under ambient (aCO2) and elevated CO2 (eCO2). Shown are quarter-hourly treatment means over three (aCO2) and four (eCO2) days of acclimation with shaded areas depicting ± standard deviation (n = 6 seedlings per treatment).